Lymphatic metastasis is the main dissemination pathway in many solid tumors. The prerequisite for this process is generation of new lymphatic vessels accessible to tumor cells. The formation of new lymphatic vessels, i.e., lymphangiogenesis, is thought to be induced primarily by two factors, VEGF-C or VEGF-D. We recently discovered that an antibody against another member of the VEGF family, VEGF-A, inhibits both tumor lymphangiogenesis and lymphatic metastasis without affecting expression of VEGF-C or VEGF-D. This observation identifies VEGF-A, a potent angiogenic factor, as a regulator of tumor lymphangiogenesis, suggesting that anti-VEGF-A therapies could be useful for preventing lymphatic metastasis in cancer patients. This finding also provides a unique opportunity to better understand the process of lymphangiogenesis and its relevance to metastatic spread. Our in vivo data showed that anti-VEGF-A treatment suppressed expression of three pro- lymphangiogenic mediators: angiopoietin-2 (Ang-2), its receptor Tie-2 and VEGFR-3, a main receptor transmitting VEGF-C signals. The functional association among these mediators and their dependence on VEGF-A is strongly supported by both literature reports and our preliminary data. However, the mechanisms underlying VEGF-A induced and Ang-2 mediated regulation of lymphangiogenesis are largely unknown. We hypothesize that the main VEGF-A-dependent events that regulate lymphangiogenesis are: 1) transcriptional up- regulation of Ang-2, which subsequently increases expression of VEGFR-3 in lymphatic endothelium through Tie-2 activation; and 2) autocrine amplification of its own receptor in lymphatics, VEGFR-2, known to functionally enhance VEGFR-3 signaling. By increasing a number of VEGF-C receptors and enhancing their transduction activity, these VEGF-A and Ang-2 dependent events pre-sensitize lymphatics to VEGF-C and accelerate the translation of VEGF-C signals into robust formation of the new lymphatic vessels. To test these hypotheses, we propose to determine: 1) a regulatory effect of VEGF-A on the expression of VEGFR-2 in lymphatic endothelial cells in vitro; 2) a regulatory effect of Ang-2 on the expression of Tie-2 and VEGFR-3 in lymphatic endothelial cells in vitro; and 3) Ang-2 mediated, VEGF-A-independent, regulation of tumor lymphangiogenesis in breast carcinoma model in vivo. Unraveling these mechanistic details is crucially important for optimizing the existing anti-VEGF-A targeted strategy and for identifying new targets to counteract lymphatic metastasis in cancer patients. We propose to delineate the molecular events mediating inhibition of tumor lymphatics by anti-VEGF-A therapy in experimentally defined in vivo and in vitro models. The significance of these studies is two-fold: 1) they will establish a new paradigm of cross- talk among angiogenic and lymphangiogenic mediators, which would lead to a novel understanding of the formation of the lymphatic vessels in health and disease; and 2) they will define specific roles of VEGF-A and Ang-2 in the regulation of tumor lymphangiogenesis, thus providing a strong impetus for applying VEGF-A and Ang-2 inhibitors to cancer patients with a high risk of lymphatic metastasis. Because metastasis is a primary cause of mortality from cancer, these studies have the potential to significantly improve health outcomes in a large number of cancer patients. [unreadable] [unreadable] [unreadable]